- Email: [email protected]
Heavy metal levels and esterase variations between metal-exposed and unexposed duckweed Lemna minor: field and laboratory studies
Environment International 30 (2004) 811 – 814 www.elsevier.com/locate/envint
Heavy metal levels and esterase variations between metal-exposed and unexposed duckweed Lemna minor: field and laboratory studies Suman Mukherjee a, Swati Mukherjee a, P. Bhattacharyya b,*, A.K. Duttagupta a b
a Genetics Laboratory, Department of Zoology, Calcutta University, 35 B.C. Road, Calcutta-700019, West Bengal, India National Bureau of Soil Survey and Land Use Planning, Block-DK, Sector-II, Salt Lake City, Calcutta-700091, West Bengal, India
Received 8 September 2003; accepted 29 January 2004
Abstract Environmental homogeneity is being continuously disturbed and affected by artificially introduced loads of chemical toxicants that also include heavy metals. The Tiljala wetlands of the eastern fringe of Calcutta, West Bengal (India) are a virtual sink for the deposition of urban and industrial wastes that get admixed with the aquatic environment. We have selected Lemna minor (duckweed), as a representative of the biota surviving therein for the present study. Concentrations of lead, cadmium, chromium, zinc, copper and mercury in the fronds of Lemna were measured to peep into the range of input of heavy metals in the duckweed subjects. Natural unexposed population of duckweed from a domestic pond in Batanagar area, 24 Parganas, West Bengal (India) was also found to accumulate similar concentrations of these metals when cultured in artificially contaminated water in the laboratory. The exposed individuals also exhibited polymorphism with respect to the loci of esterase, as compared to an unexposed control plants. Therefore, the present study suggests EST variations of L. minor to be a potential biomarker of heavy metal pollution. D 2004 Elsevier Ltd. All rights reserved. Keywords: Heavy metals; Lemna minor; Esterase variations
1. Introduction The urban environment is incessantly being contaminated by the escalating industrialisation and urbanisation of almost all the human habitats. Considerable evidence has been cited that grade heavy metals as well-known pollutants in aquatic water bodies (Achyuthan et al., 2002). They act as conservative pollutants and often biomagnify in the food chain (Stanley and Bang, 1987; Sood et al., 1994). The hydrosphere is particularly susceptible to such misuse and degradation. The surface layers act as the first site of chemical interaction, which harbour high concentrations of surface-active molecules that act as heavy metal traps. To survive in the polluted environment, organisms need to adapt and to increase their tolerance to metal pollution. The tolerant organisms though always form a minor part of the population seem to give rise to the prevailing heteroge-
* Corresponding author. E-mail address: [email protected] (P. Bhattacharyya). 0160-4120/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2004.01.006
neity within and among the species. To investigate into the genetic basis of such acclimatization, we became interested in the stressed organisms of the eastern fringe of Calcutta that inhabit a number of polluted wetlands spread over an area of 250 km2. The wetlands are also known as the East Kolkata Fisherman’s Co-operative Society wetlands of Tiljala. These wetlands are virtually a sink for the deposition of the city’s wastewaters and sewage. A common species thriving in these waters are a group of floating aquatic plants of the family Lemnaceae (Lemna minor, common minor duckweed). As duckweeds float, it is especially susceptible to metal toxicants and because of its high reproductive rates, it adapts quickly to changes in the environment. Ben-Shlomo and Nevo (1988) suggested that the effects of heavy metals in vitro can be related to population data and it can also offer a biochemical basis for maintenance of isozyme polymorphisms in Palaemon elegans. Isozymes are a combination of polypeptides that are products of separate genes. Isozymes potentially exist as a number of allozymic states. Theoretically, the diversity of allozyme appears to be correlated with specific polluted
812
S. Mukherjee et al. / Environment International 30 (2004) 811–814
Table 1 Mean metal concentration of the exposed and unexposed niche along with their selected inhabitants (L. minor) Parameter
Lead
Cadmium
Chromium
Zinc
Copper
Mercury
Exposed east Calcutta, Tiljala water in mg/l Unexposed water (control) LSD at 0.05P Recommended level of metal concentration in water bodies (WHO, 1993) Exposed common duckweed in mg/kg (field) Unexposed control common duckweed in mg/kg (field) LSD at 0.05P
1.14 0.001 0.078 0.05 1.014 0.06 0.036
0.005 0.001 0.014 0.005 0.08 0.003 0.0023
0.43 0.002 0.112 1.0 0.9 0.23 0.042
0.25 0.021 0.08 5.0 6.42 4.49 0.042
0.03 0.004 0.02 0.05 1.2 0.8 0.033
0.02 0.006 0.0036 0.001 0.802 0.002 0.0084
LSD = least significant difference.
environments. The objective of this work is to study heavy metals levels and esterase variations in the metal-exposed and unexposed duckweed L. minor collected in the field and under laboratory study.
2. Materials and methods L. minor from the East Calcutta Fisherman’s Co-operative Society has been used in this study as an aquatic vascular plant model for laboratory toxicity testing. 2.1. Analysis of exposed organisms and water samples 2.1.1. Field study In the field, about 50 duckweed L. minor samples and water were collected from the exposed and unexposed sites, digested with HNO3 and HCl, and filtered to measure the heavy metal concentration of lead, cadmium, chromium, zinc and copper through Atomic Absorption Spectrophotometry (AAS) using air – acetylene flame except Hg, which was analysed by nitrous oxide – acetylene flame. (Page et al., 1982). The conditions of all aquaria samples studied were identical (temperature 24 jC; pH 7.8, constant flow of water). Same number of samples and amount of water was collected from an unexposed domestic pond (situated in a village near Batanagar, 24 PGS. South, West Bengal), analysed for heavy metals using AAS. 2.1.2. Laboratory study When unexposed duckweed populations were artificially stressed, out of 50 fronds that were added, 20 fronds survived among the natural unexposed population when metal exposed under laboratory conditions with metal
inputs. The cultured samples were grown for a period of 20 days and they were analysed for heavy metals and EST variations. In the laboratory experiment, we further experimented and employed artificial heavy metals to create a metal stress environment similarly like the exposed duckweeds in the field on the unexposed organisms for 20 days to amplify the potential difference that exists between the natural and the adapted populations. Based on the pooled data from the above results, fresh 50 fronds were collected and preserved in laboratory tanks maintaining similar conditions as in the natural aquaria. Salt solutions (PbCl2, CdCl2, K2CrO4, ZnSO47H2O, CuSO45H2O, HgCl2) of trace metals were used in the culture in concentrations (1, 0.005, 0.40, 0.20, 0.03 ppm) such that it corresponds with the metal levels found in the metal exposed duckweed in the field. On these samples of waters, unexposed fronds of duckweed were grown. 2.1.3. Enzyme analysis For electrophoretic analysis a discontinuous gel matrix was prepared using polyacrylamide. In order to know the variability of EST activities of both exposed and unexposed duckweed (in the field and experimental samples), they were measured following the standard enzymatic protocols taking spectrophotometric reading at 540 nm. The duckweed fronds of each particular population were homogenized in homogenizing buffer (10 ml of 0.5M Tris – HCl with 100 Al of glycerol and 100 Al of h-mercaptoethanol). Equal amount (95 Ag) of protein from 20 individual fronds was run on resolving gel matrix in each lane. After completion of electrophoresis active protein in the gel was reacted with, a-naphthyl acetate (substrate) in phosphate buffer and stained with Fast Blue RR salt (Vallejos, 1983;
Table 2 Mean metal concentration (mg/kg) of naturally thriving L. minor populations as compared with the artificially fed plants Parameter
Lead
Cadmium
Chromium
Zinc
Copper
Mercury
Exposed common duckweed in mg/kg (field) Unexposed control common duckweed in mg/kg (field) LSD at 0.005P Exposed common duckweed in mg/kg (metal exposed under laboratory) Unexposed common duckweed in mg/kg (laboratory) LSD at 0.05P
1.014 0.06 0.036 0.801 0.02 0.01
0.08 0.003 0.0023 0.06 0.003 0.0021
0.9 0.23 0.042 0.88 0.18 0.07
6.42 4.49 0.042 5.49 3.8 0.17
1.2 0.8 0.033 1.6 0.56 0.10
0.802 0.002 0.0084 0.702 0.001 0.0015
LSD = least significant difference.
S. Mukherjee et al. / Environment International 30 (2004) 811–814
813
Brewer and Sing, 1970). The run was repeated several times with an equal sample pool for a particular set (exposed or unexposed) to eradicate errors arising due to intermingling of several other parameters on the niche in question. Statistical analyses were done by SPSS 7.5 software.
3. Results and discussion Environmental perturbations are aptly illustrated by estimates of heavy metal concentration in aquatic resources. The variation depicted in the metal concentrations with respect to the exposed and the unexposed aquatic niche and the duckweed populations is represented in Table 1. For the water samples in the field, concentrations of the six metals in the exposed pond were higher than those in the unexposed pond but only Cd, Cu and Hg were significantly ( p < 0.05) higher. All the six metal levels in the exposed pond were significantly ( p < 0.05) higher than the recommended levels for metal levels in the water bodies established by WHO (1993). For the duckweed samples in the field, the six metal levels were all significantly ( p < 0.05) higher than those in the unexposed samples. For the laboratory samples, the results showed a similar profile of heavy metal concentration as depicted in Table 2. The data serves as an important grade on biomonitoring of these niches. Submerged plants possess significant potential to bioconcentrate metals relative to their environment (Guilizzoni, 1991). In the present study, an increase in metal accumulation in the plants of L. minor by replacing the metal solutions in culture is in agreement with other reports (Sinha et al., 2003). It was observed that the enzyme activity (measured by mg of 2-napthol formed/5 h incubation/mg protein) was inhibited in the conditions prevailing due to interfering toxicants. Table 3 shows the specific activity of esterase of exposed and unexposed populations of duckweed (value taken from six readings) and it is evident that the escalated activity (percentage variation of exposed from the unexposed) of esterase was about 56.76%. The variation is Table 3 Spectrophotometric readings of nonspecific esterase (mg of 2 naphthol formed/5 h incubation/mg protein) assay Observation
1 2 3 4 5 6 Range Mean % Variation from unexposed Standard deviation
Readings Exposed (E)
Unexposed (U)
0.11 0.121 0.114 0.115 0.119 0.118 0.11 – 0.121 0.116 Exposed 56.76% from unexposed 0.0039
0.07 0.075 0.069 0.07 0.078 0.081 0.069 – 0.081 0.074
0.0049
Fig. 1. The esterase isozyme pattern of L. minor fronds (E = exposed population and U = unexposed population). The PAGE was conducted on a 7.5% gel matrix.
expressed as a percent of the difference of the exposed (E) and unexposed (U) readings {(E U)/100}. Esterase isozyme polymorphism detected in L. minor populations (Fig. 1) was represented by four zones of esterase activity—Est.1, Est.2, Est.3, and Est.4 (in the order of increasing mobility from cathodal end). Zones I, III and IV became pronounced in the exposed samples while these three zones were unseen in the laboratory control samples and unexposed samples in the field. This indicated that an occurrence of specific allozymes in the EST-I, EST-III and EST-IV could play an important role in the adaptive traits for the L. minor collected from the metal exposed environment. L. minor was tolerant and adjusted to conditions of considerable stress when grown in water supplemented with heavy metals. Among aquatic macrophytes, the role of submerged plants in relation with accumulation of metals and its toxicity has been well documented (Ingole and Bhole, 2000). In their natural niche, the population did not exhibit any signs of phytotoxicity. The organisms that breed in such a level of heavy metal, that is where majority of the population cannot sustain, must be different from those who succumb (Sathyanathan, 1998). We found that the presence Est.I, Est.III and Est.IV in the metal exposed duckweed samples of the Tiljala water could be due to adaptation of the duckweeds to the metal exposed environment. Selection of particular alleles under stress of prevalent heavy metals is the outcome of the duckweed populations that have bred and adapted themselves in such exposed niche for long time.
4. Conclusion Present findings of the changes of EST in two significant metal level environments supports the fact that adaptive mechanisms in the protein levels had been triggered (Nevo et al., 1981; De Wolf et al., 2001) in order to maintain the
814
S. Mukherjee et al. / Environment International 30 (2004) 811–814
survival of the L. minor in the metal contaminated pond. The result also indicated that Esterase variations and metal contamination of the environments are interrelated. Future allozyme polymorphism studies can serve as an important monitoring system to assess stress effects in specific polluted environments.
Acknowledgements We are grateful to Dr. Fakir Ghosh, Senior Chemist, Department of Water Investigation, Government of West Bengal, India for his valuable suggestions and assistance in providing the Atomic Absorption Spectrophotometry facility. Financial assistance provided by the West Bengal Department of Science and Technology is also gratefully acknowledged.
References Achyuthan H, Richardmohan D, Srinivasalu S, Selvaraj K. Trace metals concentrations in the sediment cores of estuary and tidal zones between Chennai and Pondicherry, along the east coast of India. Indian J Mar Sci 2002;31(2):141 – 9. Ben-Shlomo R, Nevo E. Isozyme polymorphism as monitoring of marine
environments: the interactive effect of cadmium and mercury pollution on the shrimp, Palaemon elegans. Mar Pollut Bull 1988;19:314 – 7. Brewer GJ, Sing FG. An introduction to isozyme techniques. New York: Academic Press; 1970. De Wolf H, Jordaens K, Reusens E, Blust R, Backeljau T. Esterase variation in the periwinkle Littorina littorea, along the western and eastern Scheldt estuarium. Mar Environ Res 2001;52:373 – 82. Guilizzoni P. The role of heavy metals and toxic materials in the physiological ecology of submerged macrophytes. Aquat Bot 1991;41:87 – 109. Ingole NW, Bhole AG. Bio-accumulation of chromium, nickel and zinc by water hyacinth. Pollut Res 2000;19(4):575 – 83. Nevo E, Pearl T, Beiles A, Wool D. Mercury selection of allozyme genotypes in shrimps. Experientia 1981;37:1152 – 4. Page AL, Miller RH, Keeney DR. Methods of soil analysis: Part 2. 2nd ed. Madison, WI: Soil Science Society of America; 1982. Sathyanathan B. Polluting metals. Sci Report February 1998;24 – 7. Sinha S, Bhatt K, Pandey K, Singh S, Saxena R. Interactive metal accumulation and its toxic effects under repeated exposure in submerged plant Najas indica cham. Bull Environ Contam Toxicol 2003;70:696 – 704. Sood IS, Sindhu RS, Sharma KK. Bioaccumulation of heavy metals by some aquatic plants. Pollut Res 1994;13(4):331 – 43. Stanley MP, Bang MK. The role of heavy metals in human health: environment and health: adverse health and toxic effects. New York: Academic Press; 1987. p. 367 – 409. [Chap. 11]. Vallejos CE. Enzyme activity staining. In: Orton TJ, editor. Isozymes in plant genetics and breeding. Amsterdam: Elsevier; 1983. p. 469 – 516. [Part A]. World Health Organization. Guidelines for Drinking Water Quality, vols. 1 and 2. Geneva; 1993.
Heavy metal levels and esterase variations between metal-exposed and unexposed duckweed Lemna minor: field and laboratory studies Suman Mukherjee a, Swati Mukherjee a, P. Bhattacharyya b,*, A.K. Duttagupta a b
a Genetics Laboratory, Department of Zoology, Calcutta University, 35 B.C. Road, Calcutta-700019, West Bengal, India National Bureau of Soil Survey and Land Use Planning, Block-DK, Sector-II, Salt Lake City, Calcutta-700091, West Bengal, India
Received 8 September 2003; accepted 29 January 2004
Abstract Environmental homogeneity is being continuously disturbed and affected by artificially introduced loads of chemical toxicants that also include heavy metals. The Tiljala wetlands of the eastern fringe of Calcutta, West Bengal (India) are a virtual sink for the deposition of urban and industrial wastes that get admixed with the aquatic environment. We have selected Lemna minor (duckweed), as a representative of the biota surviving therein for the present study. Concentrations of lead, cadmium, chromium, zinc, copper and mercury in the fronds of Lemna were measured to peep into the range of input of heavy metals in the duckweed subjects. Natural unexposed population of duckweed from a domestic pond in Batanagar area, 24 Parganas, West Bengal (India) was also found to accumulate similar concentrations of these metals when cultured in artificially contaminated water in the laboratory. The exposed individuals also exhibited polymorphism with respect to the loci of esterase, as compared to an unexposed control plants. Therefore, the present study suggests EST variations of L. minor to be a potential biomarker of heavy metal pollution. D 2004 Elsevier Ltd. All rights reserved. Keywords: Heavy metals; Lemna minor; Esterase variations
1. Introduction The urban environment is incessantly being contaminated by the escalating industrialisation and urbanisation of almost all the human habitats. Considerable evidence has been cited that grade heavy metals as well-known pollutants in aquatic water bodies (Achyuthan et al., 2002). They act as conservative pollutants and often biomagnify in the food chain (Stanley and Bang, 1987; Sood et al., 1994). The hydrosphere is particularly susceptible to such misuse and degradation. The surface layers act as the first site of chemical interaction, which harbour high concentrations of surface-active molecules that act as heavy metal traps. To survive in the polluted environment, organisms need to adapt and to increase their tolerance to metal pollution. The tolerant organisms though always form a minor part of the population seem to give rise to the prevailing heteroge-
* Corresponding author. E-mail address: [email protected] (P. Bhattacharyya). 0160-4120/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2004.01.006
neity within and among the species. To investigate into the genetic basis of such acclimatization, we became interested in the stressed organisms of the eastern fringe of Calcutta that inhabit a number of polluted wetlands spread over an area of 250 km2. The wetlands are also known as the East Kolkata Fisherman’s Co-operative Society wetlands of Tiljala. These wetlands are virtually a sink for the deposition of the city’s wastewaters and sewage. A common species thriving in these waters are a group of floating aquatic plants of the family Lemnaceae (Lemna minor, common minor duckweed). As duckweeds float, it is especially susceptible to metal toxicants and because of its high reproductive rates, it adapts quickly to changes in the environment. Ben-Shlomo and Nevo (1988) suggested that the effects of heavy metals in vitro can be related to population data and it can also offer a biochemical basis for maintenance of isozyme polymorphisms in Palaemon elegans. Isozymes are a combination of polypeptides that are products of separate genes. Isozymes potentially exist as a number of allozymic states. Theoretically, the diversity of allozyme appears to be correlated with specific polluted
812
S. Mukherjee et al. / Environment International 30 (2004) 811–814
Table 1 Mean metal concentration of the exposed and unexposed niche along with their selected inhabitants (L. minor) Parameter
Lead
Cadmium
Chromium
Zinc
Copper
Mercury
Exposed east Calcutta, Tiljala water in mg/l Unexposed water (control) LSD at 0.05P Recommended level of metal concentration in water bodies (WHO, 1993) Exposed common duckweed in mg/kg (field) Unexposed control common duckweed in mg/kg (field) LSD at 0.05P
1.14 0.001 0.078 0.05 1.014 0.06 0.036
0.005 0.001 0.014 0.005 0.08 0.003 0.0023
0.43 0.002 0.112 1.0 0.9 0.23 0.042
0.25 0.021 0.08 5.0 6.42 4.49 0.042
0.03 0.004 0.02 0.05 1.2 0.8 0.033
0.02 0.006 0.0036 0.001 0.802 0.002 0.0084
LSD = least significant difference.
environments. The objective of this work is to study heavy metals levels and esterase variations in the metal-exposed and unexposed duckweed L. minor collected in the field and under laboratory study.
2. Materials and methods L. minor from the East Calcutta Fisherman’s Co-operative Society has been used in this study as an aquatic vascular plant model for laboratory toxicity testing. 2.1. Analysis of exposed organisms and water samples 2.1.1. Field study In the field, about 50 duckweed L. minor samples and water were collected from the exposed and unexposed sites, digested with HNO3 and HCl, and filtered to measure the heavy metal concentration of lead, cadmium, chromium, zinc and copper through Atomic Absorption Spectrophotometry (AAS) using air – acetylene flame except Hg, which was analysed by nitrous oxide – acetylene flame. (Page et al., 1982). The conditions of all aquaria samples studied were identical (temperature 24 jC; pH 7.8, constant flow of water). Same number of samples and amount of water was collected from an unexposed domestic pond (situated in a village near Batanagar, 24 PGS. South, West Bengal), analysed for heavy metals using AAS. 2.1.2. Laboratory study When unexposed duckweed populations were artificially stressed, out of 50 fronds that were added, 20 fronds survived among the natural unexposed population when metal exposed under laboratory conditions with metal
inputs. The cultured samples were grown for a period of 20 days and they were analysed for heavy metals and EST variations. In the laboratory experiment, we further experimented and employed artificial heavy metals to create a metal stress environment similarly like the exposed duckweeds in the field on the unexposed organisms for 20 days to amplify the potential difference that exists between the natural and the adapted populations. Based on the pooled data from the above results, fresh 50 fronds were collected and preserved in laboratory tanks maintaining similar conditions as in the natural aquaria. Salt solutions (PbCl2, CdCl2, K2CrO4, ZnSO47H2O, CuSO45H2O, HgCl2) of trace metals were used in the culture in concentrations (1, 0.005, 0.40, 0.20, 0.03 ppm) such that it corresponds with the metal levels found in the metal exposed duckweed in the field. On these samples of waters, unexposed fronds of duckweed were grown. 2.1.3. Enzyme analysis For electrophoretic analysis a discontinuous gel matrix was prepared using polyacrylamide. In order to know the variability of EST activities of both exposed and unexposed duckweed (in the field and experimental samples), they were measured following the standard enzymatic protocols taking spectrophotometric reading at 540 nm. The duckweed fronds of each particular population were homogenized in homogenizing buffer (10 ml of 0.5M Tris – HCl with 100 Al of glycerol and 100 Al of h-mercaptoethanol). Equal amount (95 Ag) of protein from 20 individual fronds was run on resolving gel matrix in each lane. After completion of electrophoresis active protein in the gel was reacted with, a-naphthyl acetate (substrate) in phosphate buffer and stained with Fast Blue RR salt (Vallejos, 1983;
Table 2 Mean metal concentration (mg/kg) of naturally thriving L. minor populations as compared with the artificially fed plants Parameter
Lead
Cadmium
Chromium
Zinc
Copper
Mercury
Exposed common duckweed in mg/kg (field) Unexposed control common duckweed in mg/kg (field) LSD at 0.005P Exposed common duckweed in mg/kg (metal exposed under laboratory) Unexposed common duckweed in mg/kg (laboratory) LSD at 0.05P
1.014 0.06 0.036 0.801 0.02 0.01
0.08 0.003 0.0023 0.06 0.003 0.0021
0.9 0.23 0.042 0.88 0.18 0.07
6.42 4.49 0.042 5.49 3.8 0.17
1.2 0.8 0.033 1.6 0.56 0.10
0.802 0.002 0.0084 0.702 0.001 0.0015
LSD = least significant difference.
S. Mukherjee et al. / Environment International 30 (2004) 811–814
813
Brewer and Sing, 1970). The run was repeated several times with an equal sample pool for a particular set (exposed or unexposed) to eradicate errors arising due to intermingling of several other parameters on the niche in question. Statistical analyses were done by SPSS 7.5 software.
3. Results and discussion Environmental perturbations are aptly illustrated by estimates of heavy metal concentration in aquatic resources. The variation depicted in the metal concentrations with respect to the exposed and the unexposed aquatic niche and the duckweed populations is represented in Table 1. For the water samples in the field, concentrations of the six metals in the exposed pond were higher than those in the unexposed pond but only Cd, Cu and Hg were significantly ( p < 0.05) higher. All the six metal levels in the exposed pond were significantly ( p < 0.05) higher than the recommended levels for metal levels in the water bodies established by WHO (1993). For the duckweed samples in the field, the six metal levels were all significantly ( p < 0.05) higher than those in the unexposed samples. For the laboratory samples, the results showed a similar profile of heavy metal concentration as depicted in Table 2. The data serves as an important grade on biomonitoring of these niches. Submerged plants possess significant potential to bioconcentrate metals relative to their environment (Guilizzoni, 1991). In the present study, an increase in metal accumulation in the plants of L. minor by replacing the metal solutions in culture is in agreement with other reports (Sinha et al., 2003). It was observed that the enzyme activity (measured by mg of 2-napthol formed/5 h incubation/mg protein) was inhibited in the conditions prevailing due to interfering toxicants. Table 3 shows the specific activity of esterase of exposed and unexposed populations of duckweed (value taken from six readings) and it is evident that the escalated activity (percentage variation of exposed from the unexposed) of esterase was about 56.76%. The variation is Table 3 Spectrophotometric readings of nonspecific esterase (mg of 2 naphthol formed/5 h incubation/mg protein) assay Observation
1 2 3 4 5 6 Range Mean % Variation from unexposed Standard deviation
Readings Exposed (E)
Unexposed (U)
0.11 0.121 0.114 0.115 0.119 0.118 0.11 – 0.121 0.116 Exposed 56.76% from unexposed 0.0039
0.07 0.075 0.069 0.07 0.078 0.081 0.069 – 0.081 0.074
0.0049
Fig. 1. The esterase isozyme pattern of L. minor fronds (E = exposed population and U = unexposed population). The PAGE was conducted on a 7.5% gel matrix.
expressed as a percent of the difference of the exposed (E) and unexposed (U) readings {(E U)/100}. Esterase isozyme polymorphism detected in L. minor populations (Fig. 1) was represented by four zones of esterase activity—Est.1, Est.2, Est.3, and Est.4 (in the order of increasing mobility from cathodal end). Zones I, III and IV became pronounced in the exposed samples while these three zones were unseen in the laboratory control samples and unexposed samples in the field. This indicated that an occurrence of specific allozymes in the EST-I, EST-III and EST-IV could play an important role in the adaptive traits for the L. minor collected from the metal exposed environment. L. minor was tolerant and adjusted to conditions of considerable stress when grown in water supplemented with heavy metals. Among aquatic macrophytes, the role of submerged plants in relation with accumulation of metals and its toxicity has been well documented (Ingole and Bhole, 2000). In their natural niche, the population did not exhibit any signs of phytotoxicity. The organisms that breed in such a level of heavy metal, that is where majority of the population cannot sustain, must be different from those who succumb (Sathyanathan, 1998). We found that the presence Est.I, Est.III and Est.IV in the metal exposed duckweed samples of the Tiljala water could be due to adaptation of the duckweeds to the metal exposed environment. Selection of particular alleles under stress of prevalent heavy metals is the outcome of the duckweed populations that have bred and adapted themselves in such exposed niche for long time.
4. Conclusion Present findings of the changes of EST in two significant metal level environments supports the fact that adaptive mechanisms in the protein levels had been triggered (Nevo et al., 1981; De Wolf et al., 2001) in order to maintain the
814
S. Mukherjee et al. / Environment International 30 (2004) 811–814
survival of the L. minor in the metal contaminated pond. The result also indicated that Esterase variations and metal contamination of the environments are interrelated. Future allozyme polymorphism studies can serve as an important monitoring system to assess stress effects in specific polluted environments.
Acknowledgements We are grateful to Dr. Fakir Ghosh, Senior Chemist, Department of Water Investigation, Government of West Bengal, India for his valuable suggestions and assistance in providing the Atomic Absorption Spectrophotometry facility. Financial assistance provided by the West Bengal Department of Science and Technology is also gratefully acknowledged.
References Achyuthan H, Richardmohan D, Srinivasalu S, Selvaraj K. Trace metals concentrations in the sediment cores of estuary and tidal zones between Chennai and Pondicherry, along the east coast of India. Indian J Mar Sci 2002;31(2):141 – 9. Ben-Shlomo R, Nevo E. Isozyme polymorphism as monitoring of marine
environments: the interactive effect of cadmium and mercury pollution on the shrimp, Palaemon elegans. Mar Pollut Bull 1988;19:314 – 7. Brewer GJ, Sing FG. An introduction to isozyme techniques. New York: Academic Press; 1970. De Wolf H, Jordaens K, Reusens E, Blust R, Backeljau T. Esterase variation in the periwinkle Littorina littorea, along the western and eastern Scheldt estuarium. Mar Environ Res 2001;52:373 – 82. Guilizzoni P. The role of heavy metals and toxic materials in the physiological ecology of submerged macrophytes. Aquat Bot 1991;41:87 – 109. Ingole NW, Bhole AG. Bio-accumulation of chromium, nickel and zinc by water hyacinth. Pollut Res 2000;19(4):575 – 83. Nevo E, Pearl T, Beiles A, Wool D. Mercury selection of allozyme genotypes in shrimps. Experientia 1981;37:1152 – 4. Page AL, Miller RH, Keeney DR. Methods of soil analysis: Part 2. 2nd ed. Madison, WI: Soil Science Society of America; 1982. Sathyanathan B. Polluting metals. Sci Report February 1998;24 – 7. Sinha S, Bhatt K, Pandey K, Singh S, Saxena R. Interactive metal accumulation and its toxic effects under repeated exposure in submerged plant Najas indica cham. Bull Environ Contam Toxicol 2003;70:696 – 704. Sood IS, Sindhu RS, Sharma KK. Bioaccumulation of heavy metals by some aquatic plants. Pollut Res 1994;13(4):331 – 43. Stanley MP, Bang MK. The role of heavy metals in human health: environment and health: adverse health and toxic effects. New York: Academic Press; 1987. p. 367 – 409. [Chap. 11]. Vallejos CE. Enzyme activity staining. In: Orton TJ, editor. Isozymes in plant genetics and breeding. Amsterdam: Elsevier; 1983. p. 469 – 516. [Part A]. World Health Organization. Guidelines for Drinking Water Quality, vols. 1 and 2. Geneva; 1993.